1. Field of the Invention
The present invention relates to the methods of manufacturing five-carbon aldo- and keto-sugars and sugar alcohols by fermentation in recombinant hosts. Especially, the invention is directed to recombinant hosts that have been engineered to enhance the production of the pentose phosphate pathway intermediates, or the production of one or more of xylitol, D-arabitol, D-arabinose, D-lyxose, ribitol, D-ribose, D-ribulose, D-xylose, and/or D-xylulose, and to methods of manufacturing the same using such hosts.
2. Background of the Invention
Five-carbon sugars and five-carbon sugar alcohols have numerous uses as sweeteners. For example, xylitol is widely used as a non-cariogenic alternative sweetener. D-ribulose and D-xylulose, as well as sugar alcohols other than xylitol, also have potential as sweeteners in the form of free monosaccharides or as components of oligosaccharides. In that regard, glucosyl-xylulose, a close structural analog of sucrose, can be easily synthesized from sucrose and D-xylulose (Kitaoka, K., et al., Oyo Toshitsu Kagaku 41(2):165–72 (1994)).
Five-carbon sugars and five-carbon sugar alcohols are also useful for the organic and enzymatic synthesis of pharmaceuticals, functional food ingredients, etc. D-arabinose and D-lyxose are both structurally very close to D-ribose (the natural sugar constituent of nucleosides/nucleotides) and are components of many drugs and drug formulations.
Five carbon sugars and sugar alcohols are useful as carbon sources for the growth of microorganisms such as bacteria and fungi. Additionally, they are useful as biochemical reagents in laboratory assays of the enzymes that use such five carbon sugars and sugar alcohols as substrates, and as standards in the chromatographic analysis of sugars and sugar alcohols.
A sugar is said to be “naturally produced” if it is capable of being enzymatically synthesized by a non-recombinant microbial or animal host. The precursors of naturally produced five-carbon sugars and their corresponding alcohols are often the pentose phosphate pathway (PPP) sugar intermediates. These intermediates, in their 5-phosphorylated or unphosphorylated form, are valuable in and of themselves as chemical precursors of other various useful compounds. These include, for example, nucleotides and riboflavin (derived from the PPP metabolite D-ribose 5-phosphate), and folate, ubiquinone as well as various aromatic amino acids (derived from the PPP metabolite D-erythrose 4-phosphate). These amino acids are in turn precursors for flavonoids and alkaloids. Consequently, methods and hosts that increase the conversion of a raw material such as a hexose sugar into a desired PPP sugar intermediate such as ribose-5-P, ribulose-5-P or xylulose-5-P, and thus also enhance production of a desired downstream metabolite, would be of significant economical value. These compounds can be extracted or isolated or used in vivo or in vitro as is or as precursors in further metabolic/or chemical reactions to manufacture useful products.
U.S. Pat. No. 5,798,237 (Picataggio, S. K. et al.) reports a recombinant Lactobacillus that has been genetically engineered with xylose isomerase and xylulokinase genes to impart the ability to ferment lignocellulosic biomass that contains xylose to lactic acid.
Jeffries et al. have reported the genetic engineering of xylose fermentation in yeast in order to provide for the efficient production of ethanol from xylose. Jeffries, T. W. et al., “Genetic Engineering of Xylose Fermentation in Yeasts,” See: calvin.biotech.wisc.edu/jeffries/bioprocessing/xoferm/xoferm.html. Such yeast were identified by their ability to direct carbon flow from the five carbon sugar xylose into the two carbon endproducts alcohol, ethanol, most likely via a pathway that involved the PPP transketolase enzyme acting in a direction that promoted carbon flux away from PPP intermediate accumulation.
Aristidou, A. et al., WO 99/46363 reported that yeast in which the coupling of pyridine nucletide-linked dehydrogenase reactions had been improved by overexpression of NAD glutamate dehydrogenase or malic enzyme not only exhibited a more efficient production of ethanol from xylose but also had an enhanced production of xylitol from xylose.
However, little has been done with regard to modifying microorganisms in the opposite direction, to redirect carbon flow away from glycolysis or away from ethanol production and into the PPP, with accumulation of PPP intermediates and sugars or sugar alcohols derived therefrom. For example, U.S. Pat. No. 5,281,531 (Miyagawa, K. et al.) reports a method of producing D-ribose in a Bacillus host in which the gluconate operon (which encodes the proteins involved in gluconate uptake and metabolism) is partly or wholly modified so as to highly express the gluconate operon. Especially, the gntR gene is deleted or inactivated and the promoter is replaced with another.
D-ribose has been produced from glucose by fermentation with Bacillus subtilis (U.S. Pat. No. 3,607,648). Methods for the production of D-xylulose and D-ribulose by fermentation of glucose with some bacteria isolated from nature have also been described (Canadian patent 840981).
U.S. Pat. No. 3,970,522 (Sasajima, K.-I. et al.) report the production of D-ribose in a strain of Bacillus that has high 2-deoxyglucose oxidizing activity. In one strain, the Bacillus also lacks at least one of transketolase and D-ribulose phosphate 3-epimerase.
Onishi et al. have developed a multi-stage process for the production of xylitol wherein glucose is first fermented with an osmophilic yeast into D-arabitol. Using a different strain D-arabitol is then converted in a second fermentation into D-xylulose. Lastly, using a third strain and in a third fermentation, D-xylulose is reduced to xylitol by fermentation (Onishi, H. and Suzuki, T., Appl. Microbiol. 18:1031–1035 (1969)).
Harkki et al. have developed a one-stage fermentation process to convert glucose into xylitol and were the first to suggest directly modifying the PPP for the production of xylitol from glucose in a single host (U.S. Pat. No. 5,631,150).
Many of the above microbiological methods use strains of bacteria isolated from nature. Most teach no methods of further improving the native abilities the of microorganisms for the production of such sugars or sugar alcohols, or for broadening the spectrum of useful products produced by the fermentation. While the work of Harkki et al. (U.S. Pat. No. 5,631,150) describes some methods of metabolically engineering hosts and methods for the production of xylitol in such hosts, especially by over-expression of the genes of the oxidative branch of PPP, nevertheless, clearly, additional methods for enhancing the metabolic flux through the PPP would be beneficial for production of five carbon sugars as well as any PPP-derived product or product precursor.